To Purify Homocysteine Methyl Transfer Enzyme And Determine Activity

Objective1 To culture escherichia coli which contain exogenous genes of homocysteine methyltransferase and collect supernatant which contain homocysteine methyltransferase.2 To separate and purify the homocysteine methyltransferase3 To judge whether the purified homocysteine methyltransferase has activity or not; determining its activity preliminarily.Methods1. The escherichia coli which containing exogenous genes of homocysteine methyltransferase were inoculated in the blood agar. the blood agar were culture at 37℃overnight, then the escherichia coli were inoculated to Medium LB which contain ampicillin and cultured to mid-log phase in shake at 37℃. After adding IPTG to Medium LB for 8 hours, the liquid were centrifuged for obtaining precipitation. Precipitation were dissolved with buffer, then mixed with electric mixer(5000 r/min,2min) to destroy bacteria. The supernatant obtained after centrifugation (15000g,15min) were loaded on to the Sephadex CL-6B column, the DEAE Sepharose Fast Flow column and the Sephadex G-75 column for obtaining the purified homocysteine methyltransferase.2. To verify whether protein peaks after chromatography contain homocysteine methyltransferase by applying Western Blot and Coomassie brilliant blue staining, then determine roughly the molecular weight according to Mark strip.3. To determine the homocysteine concentration in serum by using Homocysteine Test Kit, replace reagent 2 with the mix liquor which is consisted of the purified homocysteine methyltransferase and glutamate dehydrogenase, then re-testing the homocysteine concentration of the same sample, at last to determine if the purified homocysteine methyltransferase has activity according to the result of two kinds of kits.Results1. The enzyme purified is what we needed through Western blot verification.2. Compared with the Western Blot and Coomassie brilliant blue staining bands on Mark, the molecular weight of homocysteine methyltransferase monomer is 45kD.3. we determined the homocysteine concentration in serum(n=20) separately by using the Homocysteine Test Kit and the kit that reagent 2 has been replaced; analyzed the result with matched t-test. Data are shown as mean±SD, the result of the Homocysteine Test Kit is 16.48±3.48, standard error is 0.77. the result of the kit that reagent 2 has been replaced is 17.19±4.10, standard error is 0.92. The correlation coefficient of the two method is 0.92, the P value of the correlation coefficient<0.05,so we concluded that the two method correlated extremely. the two method wasn't regarded significant because the P value of matched t-test>0.05.Conclusion1. The homocysteine methyltransferase was purified successfully, we established the method of purification that can be used on research and production. 2. The molecular weight of homocysteine methyltransferase that we purified monomer is 45kD.3. The homocysteine methyltransferase that we purified has activity. It can be used to detect the concentration of homocysteine.